Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images.

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FcγRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcγRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcγRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position −302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcγRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.

Serotonin [5-hydroxytryptamine (5-HT)] is a major therapeutic target of psychiatric disorders. Tryptophan hydroxylase (TPH) catalyzesthe rate-limiting reaction in the biosynthesis of 5-HT. Two isoforms (TPH1 and TPH2) having tryptophan hydroxylating activity were identified. Association studies have revealed possible TPH1 involvement in psychiatric conditions and behavioral traits. However, TPH1 mRNA was reported to be mainly expressed in the pineal gland and the periphery and to be barely detected in the brain. Therefore, contribution of TPH1 to brain 5-HT levels is not known, and the mechanisms how TPH1 possibly contributes to the pathogenesis of psychiatric disorders are not understood. Here, we show an unexpected role of TPH1 in the developing brain. We found that TPH1 is expressed preferentially during the late developmental stage in the mouse brain. TPH1 showed higher affinity to tryptophan and stronger enzyme activity than TPH2 in a condition reflecting that of the developing brainstem. Low 5-HT contents in the raphe nucleus were seen during development in New Zealand white (NZW) and SWR mice having common functional polymorphisms in the TPH1 gene. However, the 5-HT contents in these mice were not reduced in adulthood. In adult NZW and SWR mice, depression-related behavior was observed. Considering an involvement of developmental brain disturbance in psychiatric disorders, TPH1 may act specifically on development of 5-HT neurons, and thereby influence behavior later in life.

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-L-tryptophan (5-HTP)has been simply regarded as a precursor of serotonin , and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans. The mature small intestinal surface is covered with abundant finger-like villi that project into the luminal space. The apical surface of the mature intestinal epithelium is covered with dense brush border microvilli. The formation of the microvilli is regulated by the organization of individual cell membrane domains and cell:cell junctions via interaction between membrane proteins and actin cytoskeleton.Serotonin [5-hydroxytryptamine (5-HT)] has been implicated in a number of physiological and pathological functions in several peripheral organs and tissues such as liver, platelet, and immune systems.1-6 An enormous proportion of 5-HT is produced in the enterochromaffin cells and is stored in the platelets that release 5-HT in multiple peripheral organs. It has been believed that the released 5-HT essentially exerts its biological effects via 5-HT receptors on various cells in a paracrine manner. There are multiple types of 5-HT receptors that are distributed widely among endocrine, cardiovascular, immune, and gastrointestinal tissues. Receptors for 5-HT fall into one of four distinct families (5-HTR1, 5-HTR2, 5-HTR3, 5-HTR4-7), which are characterized by different signal transduction mechanisms and physiological roles. In the gut, 5-HT plays several physiological roles. For example, 5-HT has been reported to increase the rate at which enterocyte precursors proliferate, and the enhancement of enterocyte proliferation by 5-HT might be mediated by a 5-HT2 receptor. 7The biosynthesis of 5-HT is accomplished through multistep enzyme reactions. Tryptophan hydroxylase-1 (TPH-1) and TPH-2 catalyze the formation of 5-hydroxy-L-tryptophan (5-HTP) from L-tryptophan, the first and ratelimiting step in the biosynthesis of 5-HT. Subsequently, 5-HT is produced from 5-HTP by aromatic L-amino acid decarboxylase (AADC). Despite the frequent use of 5-HTP to t...

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The immune and nervous systems display considerable overlap in their molecular repertoire. Molecules originally shown to be critical for immune responses also serve neuronal functions that include normal brain development, neuronal differentiation, synaptic plasticity, and behavior. We show here that Fc␥RIIB, a low-affinity immunoglobulin G Fc receptor, and CD3 are involved in cerebellar functions. Although membranous CD3 and Fc␥RIIB are crucial regulators on different cells in the immune system, both CD3 and Fc␥RIIB are expressed on Purkinje cells in the cerebellum. Both CD3-deficient mice and Fc␥RIIB-deficient mice showed an impaired development of Purkinje neurons. In the adult, rotarod performance of these mutant mice was impaired at high speed. In the two knockout mice, enhanced paired-pulse facilitation of parallel fiberPurkinje cell synapses was shared. These results indicate that diverse immune molecules play critical roles in the functional establishment in the cerebellum.Some molecules originally shown to be critical for immune responses, such as the major histocompatibility complex (MHC) class I molecules, CD3, and semaphorin 7A (3,8,15,23), also serve neuronal functions. Based on studies of mutant mice, CD3 proved critical for the development of lateral geniculate nucleus (LGN) and long-term synaptic plasticity in the adult hippocampus (3,8).In the immune system, CD3 subunits are expressed on T cells. The T-cell receptor (TCR)-CD3 complex recognizing specific antigens bound to MHC present on antigen-presenting cells (APCs) is composed of a TCR heterodimer and CD3 polypeptides organized as dimers. The cell-cell interaction between APCs and T cells is known as an immunological synapse (5) in the mature immune system. In ␣␤ T cells, when the TCR interacts with the antigen/MHC complex, it transmits information to a signal-transducing complex consisting of two CD3 subunit dimers, CD3ε-CD3␥ and CD3ε-CD3␦, and the CD3-CD3 homodimer (10). Among CD3 subunits, CD3 is a crucial subunit having three immunoreceptor tyrosine-based activation motifs (ITAMs), whereas the remaining subunits have one ITAM (25). Tyrosine residues within these motifs are phosphorylated by src family tyrosine kinases, and then Src homology 2-containing proteins, including the tyrosine kinase ZAP70, participate in signaling (13). The signaling in ␥␦ TCRs is different from that in ␣␤ TCRs. Most ␥␦ TCRs lack CD3␦, and signal transduction by ␥␦ TCR is superior to that by ␣␤ TCR, as measured by its ability to induce calcium mobilization, extracellular signal-regulated kinase activation, and cellular proliferation (6).Fc␥RIIB is a low-affinity membrane receptor for immune complexes broadly distributed on hematopoietic cells, such as B cells, mast cells, basophils, macrophages, eosinophils, neutrophils, dendritic cells, and Langerhans cells. Fc␥RIIB negatively regulates B-cell receptor-induced signaling in B cells via the inhibitory immunoreceptor tyrosine-based inhibition motif in its cytoplasmic domain (24,30). Coengagement of the B-cell rece...

We evaluated the optimal detection angle for maximizing the signal to noise ratio (SNR) in sub-diffraction resolution photothermal microscopy. The angular dependent photothermal signal was calculated based on scattering theory using the temporally modulated Yukawa potential, and its detection angle and modulation frequency dependencies were analyzed. We verified the theoretical findings by imaging gold nanoparticles using laser diode based photothermal microscopy with balanced detection scheme. High-sensitivity (SNR ~40) photothermal biological imaging of a mouse brain was also demonstrated.